The present invention relates to novel amidino compounds, to a process for their manufacture, to pharmaceutical compositions containing them, and to their use in therapy, in particular their use as selective inhibitors of inducible nitric oxide synthase.
Nitric oxide is the endogenous stimulator of the soluble guanylate cyclase enzyme and is involved in a number of biological actions. Excess nitric oxide production is also thought to be involved in a number of conditions, including septic shock and many inflammatory diseases. The biochemical synthesis of nitric oxide from L-arginine is catalysed by the enzyme NO synthase. Many inhibitors of NO synthase have been described and proposed for therapeutic use.
More recently, it has been an object in this field to provide NO synthase inhibitors displaying selectivity for either inducible NO synthase (iNOS) or neuronal NO synthase (nNOS) over endothelial NO synthase (eNOS).
Thus WO93/13055 describes selective NO synthase inhibitors of formula 
and salts, and pharmaceutically acceptable esters and amides thereof, in which:
R1 is a C1-6 straight or branched chain alkyl group, a C2-6alkenyl group, a C2-6alkynyl group, a C3-6cycloalkyl group or a C3-6cycloalkylC1-6alkyl group;
Q is an alkylene, alkenylene or alkynylene group having 3 to 6 carbon atoms and which may optionally be substituted by one or more C1-3alkyl groups;
a group of formula xe2x80x94(CH2)pX(CH2)qxe2x80x94 where p is 2 or 3, q is 1 or 2 and X is S(O)x where x is 0, 1 or 2, O or NR2 where R2 is H or C1-6alkyl; or
a group of formula xe2x80x94(CH2)rA(CH2)sxe2x80x94 where r is 0, 1 or 2, s is 0, 1 or 2 and A is a 3 to 6 membered carbocylic or heterocyclic ring which may optionally be substituted by one or more suitable substituents such as C1-6alkyl, C1-6alkoxy, hydroxy, halo, nitro, cyano, trifluoroC1-6alkyl, amino, C1-6alkylamino or diC1-6alkylamino.
We have now found compounds falling within the scope of WO 93/13055 which as well as being selective iNOS inhibitors, display advantages including that they have a long half-life and are orally bioavailable when administered in vivo.
Therefore, according to the present invention there is provided a compound of formula (I) 
or a salt, solvate, or physiologically functional derivative thereof.
Formula (I) includes an asymmetric centre in the amino acid group, and although the natural L or (S) configuration of arginine is preferred, it is intended that formula (I) includes both (S) and (R) enantiomers either in substantially pure form or admixed in any proportions.
Thus, in the alternative, the present invention provides a compound selected from:
(R/S)-[2-(1-iminoethylamino)ethyl]-DL-homocysteine
(S)-[2-(1-iminoethylamino)ethyl]-L-homocysteine; and
(R)-[2-(1-iminoethylamino)ethyl]-D-homocysteine
and salts, solvates, and physiologically functional derivatives thereof.
In a preferred aspect, the present invention provides (S)-[2-(1-iminoethylamino)ethyl]-L-homocysteine or a salt, solvate, or physiologically functional derivative thereof. In a particularly preferred aspect, the present invention provides (S)-2-(1-iminoethylamino)ethyl]-L-homocysteine or a salt thereof.
Salts and solvates of compounds of formula (I) which are suitable for use in medicine are those wherein the counterion or associated solvent is pharmaceutically acceptable. However, salts and solvates having non-pharmaceutically acceptable counterions or associated solvents are within the scope of the present invention, for example, for use as intermediates in the preparation of other compounds of formula (I) and their pharmaceutically acceptable salts, solvates, and physiologically functional derivatives.
By the term xe2x80x9cphysiologically functional derivativexe2x80x9d is meant a chemical derivative of a compound of formula (I) having the same physiological function as the free compound of formula (I), for example, by being convertible in the body thereto. According to the present invention, examples of physiologically functional derivatives indude esters, amides, and carbamates; preferably esters and amides.
Suitable salts according to the invention include those formed with both organic and inorganic acids or bases. Pharmaceutically acceptable acid addition salts include those formed from hydrochloric, hydrobromic, sulphuric, citric, tartaric, phosphoric, lactic, pyruvic, acetic, trifluoroacetic, succinic, oxalic, fumaric, maleic, oxaloacetic, methanesulphonic, ethanesulphonic, p-toluenesulphonic, benzenesulphonic, and isethionic acids. Pharmaceutically acceptable base salts include ammonium salts, alkali metal salts such as those of sodium and potassium, alkaline earth metal salts such as those of calcium and magnesium and salts with organic bases such as dicyclohexyl amine and N-methyl-D-glucamine.
Pharmaceutically acceptable esters and amides of the compounds of formula (I) may have the acid group converted to a C1-6alkyl, aryl, aryl C1-6 alkyl, or amino acid ester or amide. Pharmaceutically accceptable amides and carbamates of the compounds of formula (I) may have an amino group converted to a C1-6alkyl, aryl, aryl C1-6 alkyl, or amino acid amide or carbamate.
As mentioned above, the compounds of formula (I) are inhibitors of NO synthase as demonstrated in the NOS inhibition assays below.
Therefore, compounds of formula (I) and their pharmaceutically acceptable salts, solvates, and physiologically functional derivatives have use in the prophylaxis and treatment of clinical conditions for which an inhibitor of NO synthase is indicated, in particular, an inhibitor of iNOS. Such conditions include inflammatory conditions, shock states, immune disorders, and disorders of gastrointestinal motility. The compounds of formula (I) and pharmaceutically acceptable salts, solvates, and physiologically functional derivatives thereof may also be of use in the prophylaxis and treatment of diseases of the central nervous system including migraine.
By shock states is meant those resulting from overproduction of NO, such as septic shock, haemorrhagic shock, traumatic shock, or shock caused by fulminant hepatic failure or by therapy with cytokines such as TNF, IL-1 and IL-2 or therapy with cytokine-inducing agents, for example 5,6-dimethylxanthenone acetic acid.
Examples of inflammatory conditions and immune disorders include those of the joint, particularly arthritis (e.g. rheumatoid arthritis, osteoarthritis, prosthetic joint failure), or the gastrointestinal tract (e.g. ulcerative colitis, Crohn""s disease, and other inflammatory bowel diseases, gastritis and mucosalI inflammation resulting from infection, the enteropathy provoked by non-steroidal antiinflammatory drugs), of the lung (e.g. adult respiratory distress syndrome, asthma, cystic fibrosis, or chronic obstructive pulmonary disease), of the heart (e.g. myocarditis), of nervous tissue (e.g. multiple sclerosis), of the pancreas (e.g. diabetes melitus and complications thereof), of the kidney (e.g. glomerulonephritis), of the skin (e.g. dermatitis, psoriasis, eczema, urticaria), of the eye (e.g. glaucoma) as well as of transplanted organs (e.g. rejection) and multi-organ diseases (e.g. systemic lupus erythematosis) and inflammatory sequelae of viral or bacterial infections.
Furthermore, there is evidence for overproduction of NO by iNOS in atherosclerosis and following hypoxic or ischaemic insults (with or without reperfusion), for example in the brain or in ischaemic heart disease.
Disorders of gastrointestinal motility include ifeus, for example post-operative ileus and ileus during sepsis.
By diseases of the central nervous system is meant those for which overproduction of NO is implicated, for example migraine, psychosis, anxiety, schizophrenia, sleep disorders, cerebral ischaemia, CNS trauma, epilepsy, multiple sclerosis, AIDS dementia, chronic neurodegenerative disease (e.g. Lewy Body Dementia, Huntington""s disease, Parkinson""s disease, or Alzheimer""s disease) and acute and chronic pain, and conditions in which non-adrenergic non-cholinergic nerve may be implicated such as priapism, obesity and hyperphagia.
Examples of acute pain include musculoskeletal pain, post operatives pain and surgical pain. Examples of chronic pain include chronic inflammatory pain (e.g. rheumatoid arthritis and osteoarthritis), neuropathic pain (e.g. post herpetic neuralgia, diabetic neuropathies associated with diabeties, trigeminal neuralgia, pain associated with functional bowel disorders, e.g. irritable bowel syndrome, non cardiac chest pain and sympathetically maintained pain) and pain associated with cancer and fibromyalgia.
Furthermore, inhibition of NO synthase may be of advantage in preventing the lymphocyte loss associated with HIV infection, in increasing the radiosensitivity of tumours during radiotherapy and in reducing tumour growth, tumour progression, angiogenesis, and metastasis.
Accordingly, the present invention provides a method for the prophylaxis or treatment of a clinical condition in a mammal, such as a human, for which an inhibitor of nitric oxide synthase, for example, an iNOS inhibitor is indicated, which comprises administration of a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, solvate, or physiologically functional derivative thereof. In particular, the present invention provides a method for the prophylaxis or treatment of an inflammatory and/or immune disorder, such as arthritis or asthma. In a preferred aspect the present invention provides a method for the prophylaxis or treatment of a clinical condition selected from arthritis, asthma, ileus, and migraine.
In the alternative, there is also provided a compound of formula (I) or a pharmaceutically acceptable salt, solvate, or physiologically functional derivative thereof for use in medical therapy, particularly, for use in the prophylaxis or treatment of a clinical condition in a mammal, such as a human, for which an inhibitor of nitric oxide synthase, for example an iNOS inhibitor, is indicated. In particular, there is provided a compound of formula (I) or a pharmaceutically acceptable salt, solvate, or physiologically functional derivative thereof for the prophylaxis or treatment of an inflammatory and/or immune disorder, such as arthritis or asthma. In a preferred aspect, there is provided a compound of formula (I) or a pharmaceutically acceptable salt, solvate, or physiologically functional derivative thereof for the prophylaxis or treatment of arthritis, asthma, ileus, and migraine.
The amount of a compound of formula (I), or a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof which is required to achieve a therapeutic effect will, of course, vary with the particular compound, the route of administration, the subject under treatment, and the particular disorder or disease being treated. The compounds of the invention may be administered orally or via injection at a dose of from 0.1 to 1500 mg/kg per day, preferably 0.1 to 500 mg/kg per day. The dose range for adult humans is generally from 5 mg to 35 g/day and preferably 5 mg to 2 g/day. Tablets or other forms of presentation provided in discrete units may conveniently contain an amount of compound of the invention which is effective at such dosage or ais a multiple of the same, for instance, units containing 5 mg to 500 mg, usually around 10 mg to 200 mg.
While it is possible for the compound of formula (I), or a pharmaceutically acceptable salt, solvate, or physiologically functional derivative thereof to be administered alone, it is preferable to present it as a pharmaceutical formulation.
Accordingly, the present invention further provides a pharmaceutical formulation comprising a compound of formula (I) or a pharmaceutically acceptable salt, solvate, or physiologically functional derivative thereof, and a pharmaceutically acceptable carrier or excipient, and optionally one or more other therapeutic ingredients.
The present invention also provides the use of a compound of formula (I), or a pharmaceutically acceptable salt, solvate, or physiologically functional derivative thereof in the manufacture of a medicament for the prophylaxis or treatment of a clinical condition for which an inhibitor of nitric oxide synthase, for example an iNOS inhibitor, is indicated, for example an inflammatory and/or immune disorder, such as arthritis or asthma. In a preferred aspect, there is provided a compound of formula (I), or a pharmaceutically acceptable salt, solvate, or physiologically functional derivative thereof in the manufacture of a medicament for the prophylaxis or treatment of a clinical condition selected from arthritis, asthma, ileus, and migraine.
Hereinafter, the term xe2x80x9cactive ingredientxe2x80x9d means a compound of formula (I), or a pharmaceutically acceptable salt, solvate, or physiologically functional derivative thereof.
The formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular), inhalation (including fine particle dusts or mists which may be generated by means of various types of metered dose pressurised aerosols, nebulisers or insufflators), rectal and topical (including dermal, buccal, sublingual and intraocular) administration although the most suitable route may depend upon for example the condition and disorder of the recipient. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.
Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.
A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.
Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multdose containers, for example sealed ampoules and vials, and may be stored in a freezeried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example saline or water-for-injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
Formulations for rectal administration may be presented as a suppository with the usual carriers such as cocoa butter or polyethylene glycol.
Formulations for topical administration in the mouth, for example buccally or sublingually, include lozenges comprising the active ingredient in a flavoured basis such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a basis such as gelatin and glycerin or sucrose an acacia.
Preferred unit dosage formulations are those containing an effective dose, as hereinbefore recited, or an appropriate fraction thereof, of the active ingredient.
It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.
According to a further aspect of the invention, there is provided a process for preparing a compound of formula (I) or a salt, solvate, or physiologically functional derivative thereof which comprises:
(i) reaction of the compound of formula (II) 
xe2x80x83or an enantiomer, a salt, or a protected derivative thereof, with a compound of formula (III) 
xe2x80x83or a salt thereof, wherein L is a leaving group, most suitably a C1-6 alkoxy group, for example ethoxy, or an alkylthio, aralkylthio or arylthio group e.g.a benzylthio, or 1- or 2-naphthylmethylthio group; followed by the following steps in any order:
(ii) optional removal of any protecting groups;
(iii) optional separation of an enantiomer from a mixture of enantiomers;
(iv) optional conversion of the product to a corresponding salt, solvate, or physiologically functional derivative thereof.
When L is C1-6 alkoxy, the reaction in step (i) above may be effected in solution at alkaline pH, for example pH 8 to 11, suitably at pH 10.5, and at a low temperature, for example xe2x88x925xc2x0 C. to 20xc2x0 C., suitably 0 to 5xc2x0 C. When L is an alkylthio, aralkylthio, or arylthio group, the reaction may be effected in an organic solvent e.g. tetrahydrofuran or a C1-4alcohol such as ethanol, at a moderate temperature e.g. 10 to 40xc2x0 C., suitably at ambient temperature.
Compounds of formula (III) and salts thereof are available commercially or may be prepared by methods of organic chemistry well known to the person skilled in the art, for example, as described by Shearer et al in Tetrahedron Letters, 1997, 38, 179-182.
Compounds of formula (II) and salts and protected derivatives thereof may be prepared from homocystine: 
or a protected derivative thereof, by cleaving the disulphide bond to form homocysteine or a protected derivative thereof, and coupling with a compound of formula (IV) 
or a protected derivative thereof, wherein L1 is a leaving group, for example halo, such as bromo, or an alkyl, aryl or aralkyl sulphonate ester, such as toluenesulphonyl.
Cleavage of the disulphide linkage of homocystine or a protected derivative thereof to form homocysteine or a protected derivative thereof may be effected by methods known to the person skilled in the art, for example, by use of sodium in liquid ammonia, dithiothreitol, or sodium borohydride.
Protected derivatives of homocysteine, eg N-t-butoxycarbonyl homocysteine t-butyl ester, may react with compounds of formula (IV) under conditions in an appropriate organic solvent (eg toluene) in a reaction mediated by a base such as 1,8-diazabicyclo[5.4.0]undec-7-ene or a similar agent which would be recognised by one skilled in the art.
Homocystine, the compounds of formula (IV) and protected derivatives thereof are commercially available or may be prepared by methods of organic chemistry well known to the person skilled in the art.
The protecting groups used in the preparation of compounds of formula (I) may be used in a conventional manner, for example, using methods described in xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d by Theodora W Green, 2nd edition (John Wiley and Sons, 1991) which also describes methods for the removal of such groups.
In the above reactions, primary amines are suitably protected using acyl groups, such as t-butoxycarbonyl or benzyloxycarbonyl groups which may be removed under acidic conditions, for example, by treatment with hydrochloric acid or hydrobromic acid, or by hydrogenolysis.
As will be appreciated by the person skilled in the art use of such protecting groups may include orthogonal protection of amino groups in the compounds of formula (II) to facilitate the selective removal of one group in the presence of another, thus enabling selective functionalisation of a single amino function. For example, a benzyloxycarbonyl group may be selectively removed by hydrogenolysis. A person skilled in the art will also appreciate other orthogonal protection strategies, available by conventional means as described in Theodora W Green (vide supra).
The enantiomeric compounds of the invention may be obtained (a) by separation of the components of the corresponding racemic mixture, for example, by means of a chiral chromatography column, enzymic resolution methods or preparing and separating suitable diastereoisomers, or (b) by direct synthesis from the appropriate chiral intermediates by the methods described above.
Optional conversion of a compound of formula (I) to a corresponding salt may conveniently be effected by reaction with the appropriate acid or base. Optional conversion of a compound of formula (I) to a corresponding solvate or physiologically functional derivative may be effected by methods known to those skilled in the art.
According to a further aspect, the present invention provides novel intermediates for the preparation of compounds of formula (I), for example: compounds of formula (II) as defined above, or an enantiomer, a salt, or a protected derivative thereof; particularly, a compound selected from:
(S)-2,7-diamino-5-thioheptanoic acid;
(S)-7N-benzyloxycarbonyl-2,7-diamino-5-thioheptanoic acid;
(R,S)-2,7-diamino-5-thioheptanoic acid;
(R,S)-7N-benzyloxycarbonyl-2,7-diamino-5-thioheptanoic acid;
(S)-2N-t-butoxycarbonyl-2,7-diamino-5-thioheptanoic acid;
(S)-2N-t-butoxycarbonyl-7N-benzyloxycarbonyl-2,7-diamino-5-thioheptanoic acid;
(S)-t-butyl-2N-t-butoxycarbonyl-7N-benzyloxycarbonyl-2,7-diamino-5-thioheptanoate;
(S)-t-butyl-2N-t-butoxycarbonyl-2,7-diamino-5-thioheptanoate;
(R,S)-2N-t-butoxycarbonyl-2,7-diamino-5-thioheptanoic acid;
(R,S)-2N-t-butoxycarbonyl-7N-benzyloxycarbonyl-2,7-diamino-5-thioheptanoic acid;
(R,S)-t-butyl-2N-t-butoxycarbonyl-7N-benzyloxycarbonyl-2,7-diamino-5-thioheptanoate; and
(R,S)-t-butyl-2N-t-butoxycarbonyl-2,7-diamino-5-thioheptanoate.
Certain protected derivatives of the compounds of formula (I) are also useful as intermediates for the preparation of compounds of formula (I); particularly a compound selected from:
(S)-2N-t-butoxycarbonyl-7N-(1-iminoethyl)-2,7-diamino-5-thioheptanoic acid;
(S)-t-Butyl-2N-t-butoxycarbonyl-7N-(1-iminoethyl)-2,7-diamino-5-thioheptanoate;
(R,S)-2N-t-butoxycarbonyl-7N-(1-iminoethyl)-2,7-diamino-5-thioheptanoic acid;
(R,S)-t-Butyl-2N-t-butoxycarbonyl-7N-(1-iminoethyl)-2,7-diamino-5-thioheptanoate;
and salts and solvates thereof.
For a better understanding of the invention, the following Examples are given by way of illustration.